CN112236447B - T cell receptor with MAGE-B2 specificity and uses thereof - Google Patents

Abstract

The present disclosure provides methods for producing MAGE-B2 specific T cells and compositions comprising engineered MAGE-B2 specific T cell receptors. Also provided are methods of treating cancer comprising administering MAGE-B2 specific T cells.

Description

T cell receptor with MAGE-B2 specificity and uses thereof

The present application claims the benefit of U.S. provisional patent application No.62/660,083 filed on date 19, 4, 2018, which is incorporated herein by reference in its entirety.

The sequence listing contained in the file named "UTFCP1372wo_st25.Txt" at 29KB (as measured in Microsoft Windows) and created at 2019, 4, 19 was filed concomitantly with the electronic submission and is incorporated herein by reference.

Technical Field

The present application relates generally to the fields of medicine and immunology. More particularly, it relates to T cell receptors that specifically recognize the melanoma-associated antigen B2 (MAGE-B2).

Background

T cell-based therapies have shown significant promise as a method for treating many cancers; unfortunately, this approach is also hampered by the lack of immunogenic antigen targets against common cancers and potential toxicity to non-cancerous tissues. These T cell-based therapies may include adoptive cell therapy (adoptive cell therapy, ACT) and/or vaccination methods that induce an anti-tumor T cell response. Cancer vaccination methods may include delivery of specific antigens using peptide, protein, DNA or RNA vaccines, or induction of anti-cancer responses using Dendritic Cell (DC) vaccines.

ACT generally involves the infusion of autologous activated tumor-specific T cells into a patient, for example, to treat cancer. ACT has elicited a therapeutic clinical response in melanoma patients. In general, to generate an effective anti-tumor T cell response, the following three steps are generally required: sensitizing and activating antigen-specific T cells, mobilizing the activated T cells to the tumor site, and recognizing and killing the tumor by the antigen-specific T cells.

The choice of target antigen is important for inducing potent antigen-specific T cells. Although several tumor-associated antigens have been identified for melanoma and a few other solid tumor malignancies, few immunogenic targets for pancreatic, ovarian, gastric, lung, cervical, breast and head and neck cancers have been identified. Lack of target antigens that are both immunogenic and tumor specific in their expression pattern, a feature necessary to effectively treat cancer and avoid substantial off-target side effects. Thus, there is an unmet medical need for new T cell-based therapies for additional target antigens for these malignancies.

Disclosure of Invention

Certain embodiments of the present disclosure provide a T Cell Receptor (TCR) capable of binding an antigenic peptide derived from melanoma-associated antigen B2 (MAGE-B2). In one embodiment, the TCR comprises a nucleotide sequence that hybridizes to SEQ ID NO:3, and a TCR a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:5, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity. In another embodiment, a TCR is provided comprising: TCR alpha polypeptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to CDR1 (SEQ ID NO: 7), CDR2 (SEQ ID NO: 9) and CDR3 (SEQ ID NO: 11), and TCR beta polypeptides comprising sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 15) and CDR3 (SEQ ID NO: 17). In some particular aspects, the TCR comprises a polypeptide having SEQ ID NO:3 and a TCR alpha polypeptide having the sequence of SEQ ID NO: 5.

In another embodiment, the TCR comprises a nucleotide sequence that hybridizes to SEQ ID NO:19, and a TCR a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:22, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity. In another embodiment, a TCR is provided comprising: TCR alpha polypeptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to CDR1 (SEQ ID NO: 23), CDR2 (SEQ ID NO: 25) and CDR3 (SEQ ID NO: 27), and TCR beta polypeptides comprising sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to CDR1 (SEQ ID NO: 29), CDR2 (SEQ ID NO: 31) and CDR3 (SEQ ID NO: 33). In some particular aspects, the TCR comprises a polypeptide having SEQ ID NO:19 and a TCR a polypeptide having the sequence of SEQ ID NO: 22.

In some aspects, the antigenic peptide is HLA-A 2-restricted. In some aspects, the antigenic peptide is HLA-A 0201, HLA-A 0202, HLA-A 0203, HLA-A 0204, or HLA-A 0205-limiting. In some particular aspects, the antigenic peptide is HLA-A x 0201-restricted.

In some aspects, the TCR is a soluble TCR lacking a transmembrane domain. In certain aspects, the TCR further comprises a detectable label and/or a therapeutic agent.

In another embodiment, a multivalent TCR complex is provided comprising a plurality of TCRs according to the embodiment (e.g., TCRs capable of binding to an antigenic peptide derived from MAGE-B2). In some aspects, the multivalent TCR comprises 2, 3, 4, or more TCRs. In certain aspects, the multivalent TCR is present in a lipid bilayer or attached to a particle. In certain aspects, the TCR is conjugated via a linker molecule.

Another embodiment provides a polypeptide comprising: and SEQ ID NO:3 and/or a TCR a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:5, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity. Another embodiment provides a polypeptide comprising: TCR alpha polypeptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to CDR1 (SEQ ID NO: 7), CDR2 (SEQ ID NO: 9) and CDR3 (SEQ ID NO: 11) and TCR beta polypeptides comprising sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 15) and CDR3 (SEQ ID NO: 17). In some particular aspects, the polypeptide comprises SEQ ID NO:3 and the TCR alpha polypeptide of SEQ ID NO: 5. In some aspects, the polypeptide comprises SEQ ID NO:3, a TCR alpha polypeptide. In certain aspects, the polypeptide comprises SEQ ID NO: 5. Also provided herein are polynucleotides encoding the polypeptides of the embodiments.

Another embodiment provides a polypeptide comprising: and SEQ ID NO:19 and/or a TCR a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:22, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity. Another embodiment provides a polypeptide comprising: TCR alpha polypeptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to CDR1 (SEQ ID NO: 23), CDR2 (SEQ ID NO: 25) and CDR3 (SEQ ID NO: 27) and TCR beta polypeptides comprising sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to CDR1 (SEQ ID NO: 29), CDR2 (SEQ ID NO: 31) and CDR3 (SEQ ID NO: 33). In some particular aspects, the polypeptide comprises SEQ ID NO:19 and the TCR a polypeptide of SEQ ID NO: 22. In some aspects, the polypeptide comprises SEQ ID NO: 19. In certain aspects, the polypeptide comprises SEQ ID NO: 22. Also provided herein are polynucleotides encoding the polypeptides of the embodiments.

In another embodiment, there is provided an expression vector comprising the TCR of the embodiment (e.g., a TCR capable of binding an antigenic peptide derived from MAGE-B2). In some aspects, the expression vector is a viral vector. In certain aspects, the viral vector is a retroviral vector or a lentiviral vector. In a further aspect, the TCR comprises a linker domain. In some aspects, the linker domain is located between the TCR a polypeptide and the TCR β polypeptide. In certain aspects, the linker domain comprises one or more cleavage sites. In some aspects, the one or more cleavage sites are Furin (Furin) cleavage sites and/or P2A cleavage sites. In some aspects, the one or more cleavage sites are separated by a spacer. In some particular aspects, the spacer is SGSG or GSG. In some aspects, the TCR a polypeptide and the TCR β polypeptide are linked by an IRES sequence.

Also provided herein are host cells engineered to express the TCRs of the embodiments (e.g., TCRs capable of binding to antigen peptides derived from MAGE-B2).

In some aspects, the cell is an immune cell. In certain aspects, the cells are isolated from umbilical cord or blood. In some aspects, the immune cells are T cells or peripheral blood lymphokines And (5) cells. In some particular aspects, the T cell is CD8 ⁺ T cells, CD4 ⁺ T cells or γδ T cells. In some aspects, the relevant signaling molecule may be attached to a TCR and, upon TCR engagement, transmit an activation signal in a non-T cell immune effector cell. In certain aspects, the cell is an NK cell, a constant NK cell (invariant NK cell), an NKT cell, a mesenchymal stem cell (mesenchymal stem cell, MSC), or an induced pluripotent stem (induced pluripotent stem, iPS) cell. In some aspects, the cells are allogeneic or autologous. Also provided herein are pharmaceutical compositions comprising the MAGE-B2 TCR-specific cell populations of the embodiments.

Also provided herein are methods for engineering MAGE-B2 specific immune cells comprising contacting the immune cells with the expression vectors of the embodiments. In some aspects, the immune cell is a T cell, a peripheral blood lymphocyte, an NK cell, a constant NK cell, or a NKT cell. In some aspects, contacting is further defined as transfection or transduction. In certain aspects, peripheral blood lymphocytes are stimulated with OKT3 and IL-2. In further aspects, the method further comprises sorting the immune cells to isolate TCR-engineered T cells, performing T cell cloning by serial dilution, and expanding the T cell clones by a rapid expansion protocol.

In another embodiment, there is provided the use of a therapeutically effective amount of a MAGE-B2 specific TCR expressing cell according to the embodiment for treating cancer. Also provided herein are compositions comprising an effective amount of MAGE-B2 specific cells according to the embodiments for use in treating cancer in a subject. In some specific aspects, the MAGE-B2 specific TCR expressing cell is a T cell.

In another embodiment, a method of treating cancer in a subject is provided, comprising administering to the subject a therapeutically effective amount of the MAGE-B2 specific cells of the embodiment (e.g., expressing a TCR capable of binding to an antigenic peptide derived from MAGE-B2). In some aspects, the MAGE-B2 specific cell is a T cell.

In certain aspects, the subject is identified as having an HLA-A2 allele, e.g., an HLA-A 0201, HLA-A 0202, HLA-A 0203, HLA-A 0204, or HLA-A 0205 allele. In certain aspects, the subject is identified as having an HLA-A x 0201 allele. In a further aspect, the method further comprises the step of subjecting the subject to lymphocyte depletion (lymphodepletion) prior to administering a therapeutically effective amount of MAGE-B2 specific T cells. In some aspects, a therapeutically effective amount of MAGE-B2 specific T cells are derived from a sample of autologous tumor infiltrating lymphocytes (tumor infiltrating lymphocyte, TIL) having anti-tumor activity. In some aspects, MAGE-B2 specific cells are administered to a subject intravenously, intraperitoneally, or intratumorally. In some particular aspects, the subject is a human. In some aspects, the method further comprises the step of administering at least one additional therapeutic agent to the subject. In certain aspects, the at least one additional therapeutic agent is selected from chemotherapy, radiation therapy, and immunotherapy. In some aspects, the at least one additional therapeutic agent is immunotherapy. In some aspects, the immunotherapy is an immune checkpoint inhibitor. In certain aspects, the immune checkpoint inhibitor inhibits an immune checkpoint protein or ligand thereof selected from the group consisting of: CTLA-4, PD-1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or adenosine A2a receptor (A2 a receptor, A2 aR). In some aspects, the immune checkpoint inhibitor inhibits PD-1 or CTLA-4.

Brief Description of Drawings

The following drawings form a part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of some specific embodiments presented herein.

Fig. 1: western blot detection of MAGE-B2 expression in lung cancer cell lines and immortalized normal human small air epithelial cells (HSAEC 1-KT and HSAEC 2-KT).

Fig. 2: production of MAGE-B2 HLA-A2 restricted peptide specific cytotoxic T lymphocytes (cytotoxic T lymphocyte, CTL). Detection of T cell populations comprising tetramers with HLA-A2 restricted MAGE-B2 epitopes (GVYDGEEHSV) (left). For CD8 ⁺ Tetramer ⁺ Sorting of groups and rapid amplificationProtocol (rapid expansion protocol, REP) allows for amplification (medium). CTL clones were generated using limiting dilution (right).

Fig. 3A to 3D: killing ability test of MAGE-B2 CTL clone. (FIG. 3A) peptide titration of T2 cells pulsed with MAGE-B2 peptide. (FIG. 3B) through ⁵¹ CTL clones from Cr release assay were cytotoxic to lung cancer cell line H2023 (HLA-A x 0201) and normal lung cell line HSAEC2-KT (HLA-A x 0201). (FIG. 3C) cytotoxicity of CTL clones against lung cancer cell lines H522, H1355, H1755 and DFC-1032. (FIG. 3D) cytotoxicity of MAGE-B2 CTL clones against the parent lung cancer cell lines PC-9 and H1573, and both cell lines in which HLA-A2 was expressed.

Fig. 4: production of MAGE-B2T cell receptor engineered T cells (TCR-T). Infection of activated allogeneic PBMCs with retrovirus (left), and the appearance of CD8 after infection ⁺ Tetramer ⁺ (in). By CD8 ⁺ Tetramer ⁺ The population was sorted and expanded to develop TCR-T cell lines (right).

Fig. 5A to B: MAGE-B2 TCR-T killing ability assay. (FIG. 5A) peptide titration assay. T2 cells pulsed with varying concentrations of MAGE-B2 peptide. (FIG. 5B) through the standard ⁵¹ MAGE-B2 TCR-T cytotoxicity against lung cancer cell line H2023 (HLA-A.times.0201) and normal lung cell line HSAEC2-KT (HLA-A.times.0201) as measured by Cr release assay.

Fig. 6: MAGE-B2 TCR-T function assay using intracellular cytokine staining (intracellular cytokine staining, ICS).

Fig. 7: representative production of MAGE-B2 specific T cell products from dendritic cell-T cells (DC-T) from a Co-culture system with healthy donor PBMC. After 2 stimulations with MB2-231 peptide pulsed DCs, small CD8 was observed in 3 wells of a 48-well plate ⁺ Tetramer ⁺ A group. The 3 positive wells were separately sorted using tetramer-directed sorting techniques and amplified with REP for 1 or 2 rounds. CD8 and tetramer staining of the final product are shown.

Fig. 8A to 8E: functional avidity of MAGE-B2 specific T cells. (FIG. 8A) 3 species at a 20:1 effector to target (E: T) ratioMAGE-B2 CTL cell lines lyse T2 cells pulsed with various concentrations of MB2-231 peptide. (FIG. 8B) lysis of 3 MAGE-B2 CTL cell lines at various E:T ratios MAGE-B2 expressing tumor cell line H2023 (HLA-A 2) ⁺ ). Normal pulmonary cell line HSAEC2-KT (MAGE-B2) ^- ，HLA-A2 ⁺ ) Is a negative control. (FIG. 8C) lysis of the 3 MAGE-B2 CTL cell lines at various E:T ratios with MAGE-B2 expression and forced HLa-A2 expression of tumor cell line H1299-A2. The parental cell line H1299 (HLA-A 2-) is the negative control. (FIGS. 8D to 8E) 3 MAGE-B2 CTL cell lines were lysed more of the tumor cell line H1395 (MAGE-B2) ⁺ ，HLA-A2 ⁺ )、H522(MAGE-B2 ⁺ ，HLA-A2 ⁺ )、H1355(MAGE-B2 ⁺ ，HLA-A2 ⁺ )、H1755(MAGE-B2 ⁺ ，HLA-A2 ⁺ ) And DFC-1032 (MAGE-B2) ⁺ ，HLA-A2 ⁺ )。

Fig. 9A to 9E: MAGE-B2 TCR-T production and functional avidity. (FIG. 9A) detection of tetramers of TCR-T prior to infection with retrovirus containing the TCR-T gene from the high-function CTL cell line MB2-231C5, after infection, and after tetramer-directed sorting and amplification. (FIG. 9B) MB2-231C5TCR-T lysis at a 20:1 effector to target (E: T) ratio was pulsed with various concentrations of MB2-231 peptide. (FIG. 9C) MB2-231C5TCR-T lytic MAGE-B2 expressing tumor cell line H2023 (HLA-A 2) ⁺ ) Normal lung cell line HSAEC2-KT (MAGE-B2) ^- ，HLA-A2 ⁺ ) Is a negative control. (FIG. 9D) MB2-231C5 TCR-T lyses tumor cell line H1299-A2 for MAGE-B2 expression and HLa-A2 forced expression at various E: T ratios. Parental cell line H1299 (HLA-A 2 ^- ) Is a negative control. (FIG. 9E) MB2-231C5 TCR-T lyses more tumor cell lines H1395 (MAGE-B2) at various E:T ratios ⁺ ，HLA-A2 ⁺ )、H522(MAGE-B2 ⁺ ，HLA-A2 ⁺ )、H1355(MAGE-B2 ⁺ ，HLA-A2 ⁺ )、H1755(MAGE-B2 ⁺ ，HLA-A2 ⁺ ) And DFC-1032 (MAGE-B2) ⁺ ，HLA-A2 ⁺ )。

Fig. 10A to 10C: functional detection of MB2-231C5 TCR-T using an Intracellular Cytokine Staining (ICS) assay. MB2-231C5 TCR-T was co-cultured with the following at e:t=10:1 ratio: with MB2-231 peptide/M26 peptide pulsed T2, tumor cell line H2023 (MAGE-B2 ⁺ ，HLA-A2 ⁺ ) Normal lung cell line HSAEC2-KT (MAGE-B2) ^- ，HLA-A2 ⁺ ) Tumor cell line H1395 (MAGE-B2) ⁺ ，HLA-A2 ⁺ )、H522(MAGE-B2 ⁺ ，HLA-A2 ⁺ )、H1299-A2(MAGE-B2 ⁺ HLA-A2 forced expression), H1299 (MAGE-B2) ⁺ ，HLA-A2 ^- )、H1355(MAGE-B2 ⁺ ，HLA-A2 ⁺ )、H1755(MAGE-B2 ⁺ ，HLA-A2 ⁺ ) And DFC-1032 (MAGE-B2) ⁺ ，HLA-A2 ⁺ ). After overnight, markers CD137, CD69, IFN-. Gamma.and TNF-. Alpha.activated downstream of the TCR pathway were detected using ICS assay. M26 peptide pulsed T2, HSAEC2-KT, H1299 served as negative controls. After co-culture with T2, H2023, H1395, H1299-A2, H1755 pulsed with MB2-231 peptide, the levels of CD137, CD69, IFN- γ and TNF- α were significantly enhanced compared to the negative control.

Detailed Description

MelanomA-Associated antigen B2 (MAGE-B2), also known as cancer/testis antigen 3.2 (UniProt No. O15479) (CT 3.2), is encoded by a gene located on the X chromosome. MAGE-B2 is expressed in testis, but not in other normal tissues, as measured by protein and RNA levels. MAGE-B2 is overexpressed in several cancers, including lung cancer, liver cancer, head and neck cancer, stomach cancer, glioblastoma, and colorectal cancer. HLA-A2 (e.g., HLA-A 0201, HLA-A 0202, HLA-A 0203, HLA-A 0204, or HLA-A 0205) restriction peptides (GVYDGEEHSV, SEQ ID NO: 1) have been eluted from ovarian cancer cells and identified (Barnea gt al, 2002). This epitope was also identified from a polypeptide group (peptidome) analysis of glioblastoma multiforme cells T98G and U-87 (shiaibman et al, 2016).

Antigen-specific CTLs were generated in this study from peripheral blood mononuclear cells (peripheral blood mononuclear cell, PBMCs) of patients recognizing endogenous presented antigens on HLA-matched allogeneic tumor cell lines using MAGE-B2 peptide epitopes. These antigen-specific CTLs stimulated by antigen-presenting cells presenting the HLA-A 2-restricted MAGE-B2 peptide have been shown to be selectively cytotoxic against lung cancer cells.

Thus, in certain aspects, the present disclosure provides TCRs that recognize and specifically bind to MAGE-B2HLA-A2 restriction epitope GVYDGEEHSV (SEQ ID NO: 1). The present disclosure also provides nucleotide sequences encoding the TCRs, and expression vectors comprising the nucleotide sequences, which can be used to modify naive T cells and produce MAGE-B2 specific T cells. The present disclosure also provides the use of MAGE-B2 specific T cells for the treatment, e.g., adoptive cell therapy, of cancer patients whose malignant cells express the MAGE-B2 antigen (e.g., HLA-A2 positive cancer patients). Antigen-specific T cells (e.g., CTLs) provided herein can be used to target solid cancers.

I. Definition of the definition

Unless the context clearly indicates otherwise, nouns without quantitative word modifications as used herein and in the appended claims denote one or more. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "a peptide" includes reference to one or more peptides and equivalents thereof (e.g., polypeptides) known to those skilled in the art.

The term "or/and" as used herein and in the appended claims means "and/or" unless explicitly indicated to mean only alternatives or alternatives are mutually exclusive.

The term "another" as used herein and in the appended claims may mean at least a second or more.

The term "about" as used herein indicates that a particular value or metric includes inherent variation associated with the means used to obtain the metric, to calculate the value, or natural variation that exists between subjects.

For a component of a solution (e.g., a formulation of one or more proteins, polymers, or small molecules), the term "substantially free" as used herein means that the formulation is not formulated to contain the component, or that such component is present only in trace amounts (e.g., as a contaminant). In certain embodiments, if the formulation contains less than 0.05% (w/w) of the component, the formulation of the molecule of interest is substantially free of the particular component. In certain embodiments, if the formulation contains less than 0.01% (w/w) of the component, the formulation of the molecule of interest is substantially free of the particular component. In certain embodiments, a formulation of a molecule of interest is substantially free of a particular component if the amount of that particular component is not detected in the formulation using standard analytical methods (e.g., UV spectrophotometry, mass spectrometry, nuclear magnetic resonance spectroscopy, etc.).

For a component of a solution or suspension (e.g., a formulation of one or more cell types, proteins, polymers, or small molecules), the term "enriched" as used herein means that the formulation is formulated to contain a higher than normal concentration or more than a positive constant number of components (e.g., a suspension of lymphocytes may be enriched for effector T lymphocytes).

The term "treatment" and variations thereof, and the like, as used herein, refers to a process of ameliorating, reducing, or otherwise alleviating symptoms of a disease or disorder in a subject, for example, by administering a therapeutic agent to the subject, or by performing a surgical, clinical, or other medical procedure on the subject.

The terms "subject" or "patient" as used herein are used interchangeably herein to refer to an individual, such as a human or non-human organism, e.g., primate, mammal or vertebrate.

The terms "therapeutically effective" or "therapeutically beneficial" and the like as used herein refer to a therapeutic agent that ameliorates, alleviates, or otherwise alleviates one or more symptoms of a disease, disorder, or condition, or is operative, clinical, or otherwise medical practice, thereby enhancing the health of a subject suffering from a disease, disorder, or condition by, for example, reducing the frequency or severity of signs or symptoms of the disease, disorder, or condition. Thus, a therapeutically effective or therapeutically beneficial cancer treatment may, for example, reduce the size of a tumor, reduce the growth rate of a tumor, reduce the likelihood of tumor spread or metastasis.

The term "pharmaceutically acceptable" or "pharmacologically acceptable" as used herein refers to pharmaceutical formulations of therapeutic agents that do not produce an adverse, allergic, or other untoward reaction when administered to a mammalian or vertebrate subject. Such formulations should be formulated in compliance with the good manufacturing practice (good manufacturing practice, GMP) standards of sterility, pyrogenicity (pyrogenicity), purity, and any other relevant standards as required by the FDA office of biological standards (FDA Office ofBiological Standards).

The term "pharmaceutically acceptable carrier" as used herein refers to any and all chemical compounds or solvents used to formulate a therapeutic agent for delivery to a mammalian or vertebrate subject, such as, for example, aqueous solvents (e.g., water, alcohol/water solutions, saline solutions, parenteral vehicles (e.g., sodium chloride, lin Geyou dextrose (Ringer's, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters (e.g., ethyl oleate)), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelators, and inert gases), isotonic agents, absorption retarders, salts, pharmaceuticals, pharmaceutical stabilizers, gels, adhesives, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, fluids, and nutritional supplements, and any combination thereof, as will be known to those of ordinary skill in the art.

The terms "unit dose," "dose," or "dose" as used herein refer to a formulation of a therapeutic agent comprising a predetermined amount of an agent suitable for administration to a mammalian or vertebrate subject that is expected to be therapeutically effective in the subject when administered by an appropriate route and according to a desired therapeutic regimen. The actual dosage of a particular therapeutic agent to be administered to a subject may be empirically determined by the health care provider based on a variety of physical and physiological parameters including, for example, the weight, age, health, and sex of the subject, the type of disease being treated, the extent of disease progression, previous or concurrent therapeutic interventions, the route of administration, and the efficacy, stability, and toxicity of the particular therapeutic agent.

MAGE-B2 TCR methods and compositions

In certain embodiments, the present disclosure provides MAGE-B2 peptide epitopes comprising sequence GVYDGEEHSV (SEQ ID NO: 1). The MAGE-B2 peptide epitope may be contacted with or used for stimulating a population of T cells to induce proliferation of T cells that recognize or bind to the MAGE-B2 peptide epitope. MAGE-B2 peptide epitopes may be administered to a subject (e.g., a human patient) to enhance the immune response of the subject against cancer. MAGE-B2 peptide epitopes may be included in active immunotherapy (e.g., cancer vaccines) or passive immunotherapy (e.g., adoptive cell therapy). Active immunotherapy involves immunization of a subject with purified tumor antigens or MAGE-B2 peptide epitopes (natural or modified). Alternatively, antigen presenting cells pulsed with MAGE-B2 peptide epitopes (or transfected with a gene encoding a tumor antigen) may be administered to a subject. The MAGE-B2 peptide epitope may be modified or comprise one or more mutations, such as, for example, substitution mutations. Adoptive cell therapy may involve administering cells to a subject, wherein the cells (e.g., cytotoxic T cells) have been sensitized to MAGE-B2 peptide epitopes in vitro.

In particular, T cells can be activated and expanded ex vivo for adoptive cell therapy within a short period of time (e.g., 6 to 8 weeks). T cells isolated from peripheral blood (e.g., CD 4) can be isolated from the cell using, for example, tetramer-directed sorting and Rapid Expansion Protocol (REP) ⁺ T cells, CD8 ⁺ T cells, γδ T cells and regulatory T cells (tregs)) and expansion of T cells. Next, a peptide or corresponding polynucleotide (e.g., full length MAGE-B2 or MAGE-B2 peptide epitope) may be loaded into HLA-A2 positive dendritic cells, lymphoblastic cell lines (lymphoblastoid cell line, LCL), PBMC or artificial antigen presenting cells (artificial antigen presenting cell, aAPC), and then co-cultured with T cells by several rounds of stimulation to generate antigen specific CTL cell lines or clones. Furthermore, the effector function and long-term persistence in vivo of these expanded antigen-specific T cells can be enhanced by manipulation of immunomodulating parameters. These CTLs are useful for adoptive cell therapy for MAGE-B2 and HLA-A2 positive cancer patients. In addition, other MAGE-B2 specific cells that may be generated from the present disclosure include NK cells, constant NK cells, NKT cells, mesenchymal Stem Cells (MSCs), and Induced Pluripotent Stem (iPS) cells. These cells may be isolated from blood or umbilical cord. The antigen-specific cells of the present disclosure may be autologous or allogeneic.

In another approach, antigen-specific cells can be generated by using the MAGE-B2 TCRs provided herein (e.g., SEQ ID NOs: 2 to 5 or 18 to 22). In this method, the TCR sequence is inserted into a vector (e.g., a retroviral or lentiviral vector) which is introduced into a host cell, such as a T cell (e.g., CD4 ⁺ T cells, CD8 ⁺ T cells, γδ T cells and tregs), NK cells, constant NK cells, NKT cells, MSCs or iPS cells to produce antigen specific cells, which can be used for adoptive cell therapy for cancer patients.

MAGE-B2 peptide epitopes and TCR sequences are provided below.

Peptide epitope: GVYDGEEHSV (SEQ ID NO: 1)

Alpha chain (TRAV 9-2 x 01F):

alpha chain

Beta chain (TRBV 15. 02F)

Beta chain

Alpha chain CDR1

Alpha chain CDR2

Alpha chain CDR3

Beta chain CDR1

Beta chain CDR2

Beta chain CDR3

MAGE-B2-231 C5 TCR sequences are provided below. The signal peptide is underlined and the variable regions are shown in italics.

Alpha chain (TRAV 10 x 01):

alpha chain

Beta chain (TRBV 11-3 x 04)

Beta chain

Alpha chain CDR1

Alpha chain CDR2

Alpha chain CDR3

Beta chain CDR1

Beta chain CDR2

Beta chain CDR3

MAGE-B2 peptides

In some aspects, the disclosure comprises MAGE-B2 peptide epitopes. The MAGE-B2 peptide epitope may have the amino acid sequence GVYDGEEHSV of the HLA-A2 restricted MAGE-B2 peptide; SEQ ID NO:1. the MAGE-B2 peptide epitope may have a sequence corresponding to SEQ ID NO:1 has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98, 99, or 100 percent sequence identity.

The MAGE-B2 peptide epitope may comprise or consist of: 7 to 35 amino acids, preferably 8 to 35 amino acid residues, and even more preferably 8 to 25 amino acids, or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids in length, or any range derivable therein. For example, in some embodiments, a MAGE-B2 peptide epitope of the present disclosure may comprise SEQ ID NO:1 or consists of a MAGE-B2 peptide epitope of 1. The antigenic peptide may comprise an immunoreactive MAGE-B2 peptide epitope, and may comprise additional sequences. Additional sequences may be derived from natural antigens and may be heterologous, and such sequences may (but need not) be immunogenic. In some embodiments, the MAGE-B2 peptide epitope may be selectively conjugated to HLA-A2, in particular HLA-A 0201, HLA-A 0202, HLA-A 0203, HLA-A 0204 or HLA-A 0205.

As will be appreciated by those skilled in the art, MHC molecules may bind peptides of different sizes, but typically do not bind full-length proteins. Although MHC class I molecules have traditionally been described as binding to peptides 8 to 11 amino acids long, it has been shown that peptides 15 amino acids long can bind to MHC class I molecules by either bulging in the middle of the binding site or extending beyond the MHC class I binding groove. As the skilled artisan will immediately appreciate, a naturally occurring full length tumor antigen (e.g., MAGE-B2) will not be available to selectively bind to MHC class II such that the antigen is internalized and T cell proliferation occurs. In general, naturally occurring full length tumor antigen proteins do not exhibit these properties and will therefore not be useful for these immunotherapeutic purposes.

In certain embodiments, the MAGE-B2 peptide epitope is immunogenic or antigenic. As shown in the examples below, the MAGE-B2 peptide epitopes of the present disclosure may promote proliferation of T cells.

The MAGE-B2 peptide epitope can be a recombinant peptide, a synthetic peptide, a purified peptide, an immobilized peptide, a detectably labeled peptide, an encapsulated peptide, or a peptide expressed via a vector (e.g., a peptide encoded by a nucleic acid in a vector comprising a heterologous promoter operably linked to the nucleic acid). In some embodiments, the synthetic MAGE-B2 peptide epitope can be administered to a subject (e.g., a human patient) to induce an immune response in the subject. Synthetic peptides may exhibit certain advantages over recombinantly expressed peptides, such as reduced risk of bacterial contamination. The MAGE-B2 peptide may also be included in a pharmaceutical composition (e.g., such as a vaccine composition) formulated for administration to a mammalian or human subject.

1. Cell penetrating peptide

In some embodiments, immunotherapy may use MAGE-B2 peptide epitopes of the present disclosure in association with cell penetrating agents, such as liposomes or cell penetrating peptides (cell penetrating peptide, CPP). Antigen presenting cells pulsed with peptides (e.g., dendritic cells) can be used to enhance anti-tumor immunity. In some embodiments, immunotherapy may use nucleic acids encoding MAGE-B2 peptide epitopes of the present disclosure, wherein the nucleic acids are delivered, for example, in viral vectors or non-viral vectors.

Cell penetrating peptides that can be covalently bound to tumor antigen specific peptides (e.g., MAGE-B2 peptides) include, for example, HIV Tat, herpes virus VP22, drosophila antennapedia (Drosophila Antennapedia) homeobox gene products, signal sequences, fusion sequences, or antimicrobial peptides I (protegrin I). Covalent binding of the peptide to the CPP can prolong presentation of the peptide by dendritic cells, thereby enhancing antitumor immunity. In some embodiments, the MAGE-B2 peptides of the present disclosure (e.g., contained within a peptide or multi-epitope string) can be covalently bound (e.g., via a peptide bond) to a CPP to produce a fusion protein. In other embodiments, the MAGE-B2 peptide epitope or nucleic acid encoding the peptide epitope may be encapsulated within or associated with a liposome (e.g., multilamellar, vesicular, or multivesicular liposome), extracellular vesicle, or exosome.

In some embodiments, cellular uptake is facilitated by attaching a lipid (e.g., stearate or myristate) to the polypeptide. It has been shown that lipidation (lipid) enhances the entry of peptides into cells. Attachment of lipid moieties is another way of the present disclosure to enhance polypeptide uptake by cells. Cellular uptake is discussed further below.

MAGE-B2 peptide epitopes of the disclosure may be included in liposomal vaccine compositions. For example, the liposome composition can be or comprise a proteoliposome composition (proteoliposomal composition).

In some embodiments, the MAGE-B2 peptide epitope can associate with a nanoparticle to form a nanoparticle-polypeptide complex. In some embodiments, the nanoparticle is a liposome or other lipid-based nanoparticle, such as a lipid-based vesicle (e.g., DOTAP: cholesterol vesicle). In other embodiments, the nanoparticle is a superparamagnetic nanoparticle based on iron oxide. In some embodiments, the nanoparticle is a semiconductor nanocrystal or a semiconductor quantum dot, both of which can be used for optical imaging. In other embodiments, the nanoparticle may be a nanoshell comprising a gold layer over a silica core.

2. Biological functional equivalents

The MAGE-B2 peptide epitopes of the disclosure may be modified to comprise amino acid substitutions, insertions, and/or deletions that do not alter their interaction with HLA class protein (e.g., HLA-A 2) binding regions, respectively. As one non-limiting example, certain amino acids in the MAGE-B2 peptides disclosed herein may be replaced with other amino acids without appreciable loss of HLA binding, as indicated by the detectably unchanged peptide binding to HLA-a 2. It is therefore contemplated that MAGE-B2 peptides disclosed herein (or nucleic acids encoding such peptides) that are modified in sequence and/or structure but have no altered biological utility or activity remain within the scope of the compositions and methods disclosed herein.

The skilled person also fully understands that inherent to the definition of biologically functionally equivalent peptides is the following concept: there are limits to the number of changes that can be made in a defined portion of a molecule while still maintaining an acceptable level of equivalent biological activity. Thus, biologically functionally equivalent peptides are defined herein as those peptides in which some (not most or all) of the amino acids may be replaced. Of course, a variety of unique peptides with different substitutions can be readily made and used in accordance with the present disclosure.

The skilled artisan will also appreciate that where certain residues (e.g., residues in a particular epitope) are shown to be particularly important for the biological or structural properties of the peptide, such residues may not typically be exchanged. This may be the case in the present disclosure, where mutations in the MAGE-B2 peptide as disclosed herein may result in loss of species specificity and in turn reduce the utility of the resulting peptide in methods for use in the present disclosure. Thus, peptides that are antigenic (e.g., specifically bind HLA-A x 0201, HLA-A x 0202, HLA-A x 0203, HLA-A x 0204, or HLA-A x 0205) and that contain conservative amino acid substitutions are understood to be encompassed in the present disclosure. Conservative substitutions are least likely to alter the activity of the protein entirely. "conservative amino acid substitutions" refer to the replacement of an amino acid with a chemically similar amino acid, i.e., the replacement of a nonpolar amino acid with another nonpolar amino acid; replacing the polar amino acid with another polar amino acid; replacement of an acidic residue with another acidic amino acid, and the like.

Amino acid substitutions (e.g., those useful for modifying the MAGE-B2 peptides disclosed herein) are generally based on the relative similarity of amino acid side chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, and the like. Analysis of the size, shape and type of amino acid side chain substituents revealed that arginine, lysine and histidine are all positively charged residues; alanine, glycine and serine all have similar dimensions; and phenylalanine, tryptophan and tyrosine all have substantially similar shapes. Thus, based on these considerations, arginine, lysine, and histidine are herein contemplated; alanine, glycine and serine; phenylalanine, tryptophan and tyrosine are defined as biological functional equivalents. In some embodiments, the mutation may enhance TCR-pMHC interaction and/or peptide-MHC binding.

The present disclosure also contemplates isoforms (isochrom) of the MAGE-B2 peptides disclosed herein. Isoforms comprise the same number and variety of amino acids as the peptides of the present disclosure, but the isoforms have different molecular structures. Isoforms contemplated by the present disclosure are those having the same properties as the peptides of the present disclosure described herein.

Non-standard amino acids can be incorporated into proteins by chemical modification of existing amino acids or by de novo synthesis of peptides disclosed herein. Non-standard amino acids refer to amino acids that differ in chemical structure from the twenty standard amino acids encoded by the genetic code.

In some embodiments, the present disclosure contemplates chemical derivatives of the MAGE-B2 peptides disclosed herein. "chemical derivative" refers to a peptide having one or more residues chemically derivatized by reaction of functional side groups and retaining biological activity and utility. Such derivatized peptides include, for example, those in which the free amino group has been derivatized to form a particular salt or p-toluenesulfonyl, benzyloxycarbonyl, t-butoxycarbonyl, chloroacetyl, formyl, acetyl, or the like has been derivatized by alkylation and/or acylation. The free carboxyl groups may be derivatized to form organic or inorganic salts, methyl and ethyl esters or other types of esters or hydrazides and preferably amides (primary or secondary amines). Chemical derivatives may include those peptides comprising one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline may be replaced with serine; ornithine may be replaced by lysine.

It should be noted that all amino acid residue sequences are represented herein by formulas oriented in their left-to-right directions from amino-terminus to carboxyl-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence represents a peptide bond to another sequence having one or more amino acid residues. The amino acids described herein are preferably in the form of the "L" isomer. However, the residues in the "D" isomer form may be replaced with any L-amino acid residue, so long as the protein retains the desired functional properties described herein.

Preferred MAGE-B2 peptides or analogs thereof preferably specifically or preferentially bind HLA-A2. Determining whether or to what extent a particular tumor antigen specific peptide or labeled peptide or analog thereof can bind to HLA-A2, and can be assessed using, for example, an in vitro assay as follows: enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, latex agglutination (latex agglutination), indirect hemagglutination assay (indirect hemagglutination assay, IHA), complement fixation (complement fixation), indirect immunofluorescence assay (FA), nephelometry (nephelometry), flow cytometry assay, chemiluminescent assay, lateral flow immunoassay, u-capture assay, mass spectrometry, particle-based assay, inhibition assay and/or affinity assay.

B. Engineered MAGE-B2 specific cells

In some embodiments, the present disclosure provides MAGE-B2 specific TCRs. The TCR may comprise SEQ ID NO:6 to 12 and/or the alpha chain CDR of SEQ ID NO:13 to 17. The TCR may comprise a sequence identical to SEQ ID NO:2 to 3 and or an alpha chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity or similarity to SEQ ID NO:4 to 5 has a β chain of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity or similarity. Also provided herein are polypeptides and polynucleotides encoding the alpha and/or beta chains of the MAGE-B2 TCRs provided herein. Also provided herein are cells engineered to express the MAGE-B2 specific TCRs provided herein, e.g., T cells, NK cells, constant NK cells, NKT cells, MSCs, or iPS cells. These non-T cell effector immune cells may express TCRs as well as CD3 molecules or other signaling domains linked to TCRs that will initiate signal transduction in these cells.

Any of a number of well-known gene transfer methods known to those skilled in the art may be used to construct the engineered immune cells. In certain embodiments, a viral vector-based gene transfer method is used to introduce nucleic acid encoding a MAGE-B2 specific TCR to construct an engineered cell. The viral vector-based gene transfer method may include lentiviral vectors, retroviral vectors, adenoviruses or adeno-associated viral vectors. In certain embodiments, non-viral vector based gene transfer methods are used to introduce nucleic acid encoding MAGE-B2 specific TCR to construct engineered cells. Vectors for TCRs may comprise an alpha chain polypeptide and a beta chain polypeptide, which may be linked by a linker domain or an IRES sequence. The linker domain may comprise one or more cleavage sites, e.g., a furin cleavage site and/or a P2A cleavage site, which may be separated by a spacer, e.g., SGSG or GSG. In certain embodiments, the non-viral vector-based gene transfer method comprises a gene editing method selected from the group consisting of: zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (transcription activator-like effector nuclease, TALENs), and clustered regularly interspaced short palindromic repeats (clustered regularly interspaced short palindromic repeat, CRISPR)/CRISPR-associated protein 9 (Cas 9) nucleases. In certain embodiments, the non-viral vector-based gene editing method comprises a transfection or transformation method selected from the group consisting of: liposome transfection, nuclear transfection, viral microvesicles, liposomes, polycations or lipid: nucleic acid conjugates, naked DNA, artificial virions and agents that enhance DNA uptake.

C. Soluble TCR and BiTE

In addition, the present disclosure provides soluble TCRs that are useful for the direct treatment of HLA-A2 positive cancer patients. Soluble bispecific T cell engagement molecules (bispecific T cell-engaging molecule, biTE) can be produced by ligating MAGE-B2 TCR with CD3 specific Fab fragments. These bispecific molecules can bind to the tumor cell surface by binding of their MAGE-B2 TCR to the peptide/HLA complex, and CD3 specific Fab fragments will crosslink, for example, TCRs on target T cells. This will result in cell activation and elimination of the target cells. Thus, these soluble bispecific TCR constructs can be used directly to treat cancer patients.

Finally, the soluble TCR can be used as a probe for diagnostic evaluation of peptides/MHC in tumor cells, or for directing therapeutic molecules to tumor sites. The soluble TCR molecules can also be labeled with a tracer (e.g., fluorescent probe or radioactive probe) and subsequently used for diagnostic evaluation of peptide/MHC presentation in tumor cells. In addition, the soluble TCR molecules can be linked to therapeutic molecules (e.g., toxins) and then directed to tumor sites to treat cancer patients.

In some embodiments, the present disclosure provides soluble TCRs, such as MAGE-B2 specific TCRs provided herein. Soluble TCRs may be used to study specific TCR-pMHC interactions, or as diagnostic tools for detecting infection or for detecting autoimmune disease markers. Soluble TCRs may be used for staining, for example for staining cells for the presence of specific peptide antigens presented in the context of MHC. Similarly, soluble TCRs can be used to deliver therapeutic agents (e.g., cytotoxic compounds or immunostimulatory compounds) to cells presenting specific antigens. Soluble TCRs may also be used to inhibit T cells, for example, those that react with autoimmune peptide antigens. In some aspects, the TCR is linked to another molecule that delivers the adjacent cell to the tumor. In other aspects, the TCR delivers a toxin, cytokine, co-stimulatory ligand, or inhibitor ligand to and directs a molecule, cell, or compound to a target cell expressing a peptide-MHC.

In some aspects, the disclosure provides a soluble T cell receptor (soluble T cell receptor, sTCR) comprising: (i) All or part of a TCR alpha chain (e.g. SEQ ID NO:2 or 3), except for its transmembrane domain, and (ii) all or part of a TCR beta chain (e.g. SEQ ID NO:4 or 5), except for its transmembrane domain, wherein (i) and (ii) each comprise a functionally variable domain and at least part of a constant domain of the TCR chain, and are linked by a disulfide bond between constant domain residues that are not found in native TCRs.

In some aspects, the soluble TCRs comprise TCR α or γ chain extracellular domains that dimerize to TCR β or δ chain extracellular domains, respectively, via a pair of C-terminal dimeric peptides (e.g., leucine zippers).

The soluble TCRs of the present disclosure may be provided in substantially pure form or as purified or isolated formulations. For example, it may be provided in a form that is substantially free of other proteins.

The various soluble TCRs of the present disclosure may be provided as multivalent complexes. Accordingly, in one aspect, the present disclosure provides multivalent TCR complexes comprising a plurality of soluble TCRs as described herein. Each of the plurality of soluble TCRs is preferably identical.

In its simplest form, a multivalent TCR complex according to the present disclosure comprises a multimer of two or three or four or more T cell receptor molecules associated (e.g., covalently or otherwise linked) with each other, preferably by a linker molecule. Suitable linker molecules include, but are not limited to, multivalent attachment molecules such as avidin, streptavidin, neutravidin, and extravidin, each having four binding sites for biotin. Thus, biotinylated TCR molecules can be formed into multimers of TCRs having multiple TCR binding sites. The number of TCR molecules in a multimer will depend on the amount of TCR associated with the amount of linker molecules used to make the multimer, and also on whether any other biotinylated molecules are present. Preferred multimers are dimeric, trimeric or tetrameric TCR complexes.

Suitable structures for use in the methods of the invention include membrane structures, such as liposomes; and solid structures, which are preferably particles, such as beads (e.g., latex beads). Other structures that can be externally coated with T cell receptor molecules are also suitable. Preferably, the structure is coated with T cell receptor multimers rather than with individual T cell receptor molecules.

In the case of liposomes, the T cell receptor molecule or multimer thereof can be attached to or otherwise associated with the membrane. Techniques for this are well known to those skilled in the art.

A tag or additional moiety (e.g., toxic or therapeutic moiety) may be included in the multivalent TCR complex of the disclosure. For example, the label or additional moiety may be included in a mixed molecular multimer. One example of such a multimeric molecule is a tetramer comprising three TCR molecules and one peroxidase molecule. This can be achieved by mixing the TCR and enzyme in a 3:1 molar ratio to produce tetrameric complexes and isolating the desired complex from any complex that does not contain the correct ratio of molecules. These mixed molecules may comprise any combination of molecules provided that the steric hindrance does not impair or significantly impair the desired function of the molecule. The localization of the binding site on the streptavidin molecule is suitable for the mixed tetramer, since steric hindrance is less likely to occur.

Alternatively or additionally, the TCRs of the present disclosure (or multivalent complexes thereof) may be associated (e.g., covalently or otherwise linked) with a therapeutic agent, which may be a toxic moiety, e.g., for use in cell killing, or an immunostimulant (e.g., interleukin or cytokine). The multivalent TCR complexes of the disclosure can have enhanced binding capacity for TCR ligands compared to non-multimeric T cell receptor heterodimers. Thus, multivalent TCR complexes according to the present disclosure are particularly useful for tracking or targeting cells presenting a particular antigen in vitro or in vivo, and are also useful as intermediates for producing additional multivalent TCR complexes for such use. Thus, the TCR or multivalent TCR complex may be provided in a pharmaceutically acceptable formulation for use in vivo.

The present disclosure also provides methods for delivering a therapeutic agent to a target cell, the methods comprising contacting a potential target cell with a TCR or multivalent TCR complex according to the present disclosure, which is specific for a TCR ligand and has a therapeutic agent associated therewith, under conditions that allow the TCR or multivalent TCR complex to attach to the target cell.

In particular, soluble TCRs or multivalent TCR complexes can be used to deliver therapeutic agents to the location of cells presenting a particular antigen. This will be useful in many situations, and in particular for tumours. Therapeutic agents may be delivered so that they exert their effect locally, but not only on the cells to which they bind. Thus, one particular strategy envisages anti-tumor molecules linked to T cell receptors or multivalent TCR complexes specific for tumor antigens.

Many therapeutic agents are useful for this purpose, such as radioactive compounds, enzymes (e.g., perforins), or chemotherapeutic agents (e.g., cisplatin). To ensure that toxic effects are carried out in the desired location, the toxin may be inside liposomes linked to streptavidin, allowing the compound to be released slowly. This will prevent damaging effects during in vivo transport and ensure that the toxin has maximum effect after the TCR binds to the relevant antigen presenting cell.

The soluble TCRs of the present disclosure may be used to modulate T cell activation by binding to specific TCR ligands and thereby inhibiting T cell activation. Autoimmune diseases involving T cell mediated inflammation and/or tissue damage (e.g., type I diabetes) would be suitable for use in this method. For this use, knowledge of the specific peptide epitope presented by the relevant pMHC is required.

Also contemplated is the use of a soluble TCR and/or multivalent TCR complex of the disclosure in the preparation of a composition for treating cancer or autoimmune disease.

Also provided are methods of treating cancer or autoimmune diseases comprising administering to a patient in need thereof an effective amount of a soluble TCR and/or multivalent TCR complex of the present disclosure.

As is common in anti-cancer and autoimmune therapies, the soluble TCRs of the present disclosure may be used in combination with other agents to treat cancer and autoimmune diseases and other related disorders found in similar patient groups.

III methods of use

In another aspect, provided herein is a method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a population of MAGE-B2 TCR-specific cells, e.g., T cells, NK cells, constant NK cells, NKT cells, MSCs, or iPS cells, produced by any of the methods provided herein. Cells can be adoptively transferred to subjects suffering from cancers from which TIL can be cultured or from which tumor antigen-specific CTLs can be produced in vitro.

Provided herein are methods for treating cancer or delaying progression of cancer in an individual comprising administering to the individual an effective amount of MAGE-B2 specific T cell therapy. Also provided herein are adoptive T cell therapies with genetically engineered TCR transduced T cells that conjugate TCRs with other biologically reactive proteins (e.g., anti-CD 3). In other embodiments, methods for treating cancer are provided that include immunizing a subject with a purified tumor antigen or an immunodominant tumor antigen specific peptide.

The MAGE-B2 peptides provided herein may be used for the development of cancer vaccines or immunogens. These peptide-specific vaccines or immunogens can be used to immunize cancer patients directly to induce an anti-tumor immune response in vivo, or to expand antigen-specific T cells in vitro with APC stimulation loaded with peptides or encoded polynucleotides. These large numbers of T cells can adoptively metastasize to patients to induce tumor regression.

Tumors that can be treated with the methods of treatment of the present invention include any malignant cell type that expresses MAGE-B2, such as those found in solid tumors or hematological tumors. Some exemplary solid tumors may include, but are not limited to, tumors of an organ selected from the group consisting of: pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate and breast. Some exemplary hematological tumors include myeloma, T or B cell malignancy, leukemia, lymphoma, blastoma, myeloma, and the like. Other examples of cancers that may be treated using the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous carcinoma), peritoneal cancer, gastric cancer (gastric cancer) or gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, renal cancer (kidney cancer), or renal cancer (renal cancer), prostate cancer, vulval cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

The cancers may specifically be of the following histological type, although they are not limited to these: tumors, malignant; cancer; cancer, undifferentiated; giant cell and spindle cell cancers; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphatic epithelial cancer; basal cell carcinoma; hair matrix cancer; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinomas; gastrinomas, malignant; bile duct cancer; hepatocellular carcinoma; combining hepatocellular carcinoma with cholangiocarcinoma; small Liang Xianai (trabecular adenocarcinoma); adenoid cystic carcinoma; adenocarcinomas among adenomatous polyps; adenocarcinomas, familial polyposis coli; solid cancer; carcinoid tumor, malignant; bronchoalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell cancer; eosinophilic cancer; eosinophilic adenocarcinoma; basophilic granulocyte cancer; clear cell adenocarcinoma; granulosa cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinomas; non-enveloped sclerotic cancers; adrenal cortex cancer; endometrial-like cancer; skin accessory cancer; apocrine adenocarcinoma; sebaceous gland cancer; cerumen adenocarcinoma; mucinous epidermoid carcinoma; cystic adenocarcinoma; papillary cyst adenocarcinoma; papillary serous cystic adenocarcinoma; mucinous cyst adenocarcinoma; mucinous adenocarcinomas; printing ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory cancer; paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; follicular membrane cytoma, malignant; granulocytoma, malignant; a male blastoma, malignancy; sertoli cell carcinoma (sertoli cell carcinoma); leydig cell tumor (leydig cell tumor), malignant; lipid cell neoplasms, malignant; paraganglioma, malignant; extramammary paraganglioma (extra-mammary paraganglioma), malignant; pheochromocytoma; vascular ball sarcoma (glomanngiosacoma); malignant melanoma; no melanotic melanoma; superficial diffuse melanoma; malignant lentigo melanomas (lentigo malignant melanoma); lentigo acromioclavis melanoma (acral lentiginous melanomas); nodular melanoma; malignant melanoma in giant pigmented nevi; epithelioid cell melanoma; blue nevi, malignant; sarcoma; fibrosarcoma; fibrohistiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; interstitial sarcoma; mixed tumor, malignant; miao Leguan mixed tumor (mullerian mixed tumor); nephroblastoma; hepatoblastoma; carcinoma sarcoma; a mesenchymal neoplasm, malignancy; brenner tumor (malignant); phylliform tumor, malignant; synovial sarcoma; mesothelioma, malignant; a vegetative cell tumor; embryonal carcinoma; teratoma, malignant; ovarian goiter, malignancy; choriocarcinoma; mesonephroma, malignancy; hemangiosarcoma; vascular endothelial tumor, malignant; kaposi's sarcoma (Kaposi's sarcoma); vascular epidermocytoma, malignant; lymphangiosarcoma; osteosarcoma; a cortical bone sarcoma; chondrosarcoma; chondroblastoma, malignant; a mesenchymal chondrosarcoma; bone giant cell tumor; ewing's sarcoma (ewing's sarcoma); odontogenic tumors, malignancy; ameloblastic osteosarcoma; enameloblastoma, malignant; ameloblastic fibrosarcoma; pineal tumor, malignancy; chordoma; glioma, malignant; ventricular tube membranoma; astrocytoma; plasmatic astrocytomas; fibroastrocytomas; astrocytoma; glioblastoma; oligodendrogliomas; oligodendroglioma; primitive neuroectoderm; cerebellar sarcoma; ganglioblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumors; meningioma, malignancy; neurofibrosarcoma; schwannoma, malignancy; granulocytoma, malignant; malignant lymphoma; hodgkin's disease; hodgkin's side granuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specific non-hodgkin lymphomas; b cell lymphoma; low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (small lymphocytic, SL) NHL; intermediate grade/follicular NHL; middle-grade diffuse NHL; advanced immunoblastic NHL; higher lymphoblastic NHL; advanced small non-dividing cell NHL; a large disease NHL (bulky disease NHL); mantle cell lymphoma; AIDS-related lymphomas; waldenstrom macroglobulinemia (Waldenstrom's macroglobulinemia); malignant histiocytohyperplasia; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic granulocytic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; prokaryotic cell leukemia; myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia (chronic lymphocytic leukemia, CLL); acute lymphoblastic leukemia (acute lymphoblastic leukemia, ALL); acute myelogenous leukemia (acute myeloid leukemia, AML) and chronic myelogenous leukemia.

In certain embodiments, the method further comprises the step of depletion of lymphocytes prior to administration of the therapeutically effective amount of the MAGE-B2TCR cell population. In certain embodiments, lymphocyte depletion comprises non-myeloablative lymphocyte depletion chemotherapy. In certain embodiments, the non-myeloablative lymphocyte depletion chemotherapy comprises administration of cyclophosphamide and fludarabine.

In certain embodiments, the method further comprises the step of administering to the subject a T cell growth factor that promotes growth and activation of autologous T cells simultaneously with or subsequent to the autologous T cells. In certain embodiments, the T cell growth factor comprises any suitable growth factor that promotes growth and activation of autologous T cells. In certain embodiments, the T cell growth factor is selected from the group consisting of Interleukins (IL) -2, IL-7, IL-15, and IL-12, and combinations thereof (e.g., IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL-2).

In certain embodiments, a therapeutically effective amount of a population of MAGE-B2 TCR-specific cells produced by any of the methods provided herein is administered to a subject intravenously, intratumorally, or intraperitoneally. The appropriate dosage for cell therapy may be determined based on the type of cancer to be treated, the severity and course of the disease, the clinical condition of the individual, the clinical history and response of the individual to the therapy, and the discretion of the attending physician.

A. Combination therapy

In certain embodiments, the methods provided herein further comprise the step of administering at least one additional therapeutic agent to the subject. Taking into account any potential toxicity, possible side effects, and any other relevant factors, all additional therapeutic agents disclosed herein are administered to a subject according to good clinical practice for each particular composition or treatment.

In certain embodiments, the additional treatment may be immunotherapy, radiation therapy, surgery (e.g., surgical excision of a tumor), chemotherapy, bone marrow transplantation, or a combination of the foregoing. The additional treatment may be a targeted treatment. In certain embodiments, the additional treatment is administered prior to the primary treatment (i.e., as an adjunct treatment). In certain embodiments, the additional treatment is administered after the primary treatment (i.e., as a neoadjuvant treatment).

In certain embodiments, the additional treatment comprises immunotherapy. In certain embodiments, the immunotherapy comprises an immune checkpoint inhibitor. In certain embodiments, the immune checkpoint inhibitor inhibits an immune checkpoint protein selected from the group consisting of: programmed cell death pathway 1 (programmed cell death pathway, PD-1/CD 279) and its ligands (PD-L1/CD 274 and PD-L2/CD 273), cytotoxic T lymphocyte-associated antigen 4 (cytotoxic T lymphocyte-associated antigen 4, CTLA-4/CD 152), lymphocyte activation gene 3 (lymphocyte-activation gene 3, LAG-3/CD 223), B and T lymphocyte attenuators (B and T lymphocyte attenuator, B and T lymphocyte attenuator, BTLA), T cell immune receptors (TIGIT) with Ig and immunoreceptor tyrosine-based inhibitory motif (immunoreceptor tyrosine-based inhibitory motif, ITIM) domains, T cell immunoglobulin domains and mucin domain 3 (T cell immunoglobulin domain and mucin domain, TIM-3/HAVcr 2), killer immunoglobulin-like receptor (killer immunoglobulin-like receptor, KIR/CD 158), T cell activated V domain immunoglobulin inhibitor (V-domain immunoglobulin suppressor of T cell activation, VISTA) and adenosine A2a receptor (A2 aR).

In certain embodiments, the immune checkpoint inhibitor is a PD-1 binding antagonist. In certain embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In certain embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab (nivolumab), pembrolizumab (pembrolizumab), and CT-011. In certain embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular portion of PDL1 or PDL2 or PD-1 binding portion fused to an immunoglobulin constant region (e.g., fc region of an immunoglobulin sequence).

In certain embodiments, the immune checkpoint inhibitor is a CTLA-4 binding antagonist. In certain embodiments, the CTLA-4 binding antagonist is an anti-CTLA-4 antibody. In certain embodiments, the anti-CTLA-4 antibody is selected from ipilimumab (ipilimumab) and tiximumab (tremelimumab).

In certain embodiments, the additional therapeutic agent comprises treatment with radiation therapy. In certain embodiments, the radiation therapy is selected from gamma rays (gamma rays), X-rays, microwaves, proton beam irradiation, ultraviolet irradiation, and direct delivery of a radioisotope to a tumor. In certain embodiments, radiation therapy comprises treatment with X-rays. In certain embodiments, the X-rays are administered at a daily dose of 50 to 200 rens over a period of three to four weeks. In certain embodiments, the X-rays are administered in a single dose of 2000 to 6000 lunqins. In certain embodiments, the radiation therapy comprises direct delivery of the radioisotope to the tumor. The dosage range of the radioisotope varies widely depending on the half-life of the isotope, the intensity and type of radiation emitted and the degree of uptake by the tumor cells, but determination of a suitable therapeutically effective dose is within the level of ordinary skill in the art.

In certain embodiments, the additional therapeutic agent comprises administration of an agent for treating side effects associated with the primary treatment (e.g., nausea, cachexia, etc.). In certain embodiments, the additional treatment comprises immunotherapy. In certain embodiments, the additional treatment comprises radiation therapy. In some embodiments, the radiation therapy comprises gamma irradiation. In certain embodiments, the additional treatment comprises surgery. In certain embodiments, the additional treatment comprises a combination of radiation therapy and surgery. In certain embodiments, the additional treatment comprises treatment with a class of chemotherapeutic agents selected from the group consisting of: alkylating agents, anthracyclines, cytoskeletal disrupting agents, epothilones, histone deacetylase inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, kinase inhibitors, nucleotide analogs and nucleotide precursor analogs, peptide antibiotics, platinum-based compounds, retinoids, vinca alkaloids, and derivatives thereof.

Additional treatments contemplated herein may be administered before, after, or concurrently with the administration of the compositions provided herein. In certain embodiments, the additional treatment is administered prior to the compositions provided herein. In certain embodiments, the additional treatment is administered after the compositions provided herein. In certain embodiments, the additional treatment is administered at one or more intervals before or after administration of the compositions provided herein. Determination of the appropriate interval of administration of additional treatments to a subject to be treated that would benefit from combination therapy is within the level of ordinary skill in the art.

B. Pharmaceutical composition

In another aspect, provided herein are pharmaceutical compositions and formulations comprising MAGE-B2 TCR specific cells and a pharmaceutically acceptable carrier.

Pharmaceutical compositions and formulations as described herein can be prepared by mixing an active ingredient (e.g., an antibody or polypeptide) of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences) ^nd The edition, 2012), in the form of an aqueous solution (e.g., physiological saline (e.g., 0.9%) and human serum albumin (e.g., 10%). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethylammonium chloride, benzalkonium chloride, benzethonium chloride, phenols, butyl alcohol or benzyl alcohol, alkyl p-hydroxybenzoates, such as methyl or propyl p-hydroxybenzoate, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); a low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG).

IV. examples

The following examples are included to demonstrate some preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 production and characterization of MAGE-B2 specific T cells

The expression of MAGE-B2 in lung cancer cell lines and immortalized normal human small air epithelial cells (HSAEC 1-KT and HSAEC 2-KT) was analyzed (FIG. 1). The MAGE-B2 protein was found to be strongly expressed in most lung cancer cell lines and was observed to be barely expressed in normal lung cell lines.

To generate MAGE-B2 specific CD8+ CTLs, dendritic cells are pulsed with RNA encoding MAGE-B2 HLA-A2 restriction epitopes. Next, T cells were stimulated with pulsed dendritic cells and CD8 was detected by flow cytometry ⁺ Tetramer. T cells were then sorted, cloned and expanded by a random expansion protocol (random expansion protocol, REP). T cells were then characterized by functional screening and then functional MAGE-B2 specific TCRs were cloned.

Thus, MAGE-B2 HLA-A2 restriction epitopes are used for the generation of MAGE-B2 specific Cytotoxic T Lymphocytes (CTLs). The naive T cells were derived from healthy HLA-A2 donors and stimulated with autologous mature dendritic cells (mature dendritic cell, mDC) pulsed with full length MAGE-B2 RNA. After two rounds of stimulation, tetramers with HLA-A2 restricted MAGE-B2 epitope (GVYDGEEHSV; SEQ ID NO: 1) were used for detection of T cell populations recognizing the epitope. Then to CD8 ⁺ Tetramer ⁺ The populations were sorted and expanded using a Rapid Expansion Protocol (REP) to generate CTL cell lines. Related CTL clones were generated using limiting dilution method. Over 99% of the cells were observed to be CD8 ⁺ And tetramer ⁺ (FIG. 2).

Next, MAGE-B2 specific T cells were tested for functional avidity. In the peptide titration assay, T2 cells were pulsed with varying concentrations of MAGE-B2 peptide (10 pg/ml to 10. Mu.g/ml) (FIG. 3A). T2 cells are used as target cells and are isolated with MAGE-BCo-culture of 2 CTL clones (E: T=20:1). Cytotoxic activity of CTL clones against lung cancer cell line H2023 (HLA-A x 0201) and normal lung cell line HSAEC2-KT (HLA-A x 0201) was measured (fig. 3B). Target cells were co-cultured with MAGE-B2 CTL clones at different E:T ratios. By standard ⁵¹ Cr release assay detects cytotoxic activity. The MAGE-B2CTL clone was observed to be cytotoxic against lung cancer cell lines, but not normal lung cell lines (FIG. 3B). In addition, HLA-A2 ⁺ The lung cancer cell lines H522, H1355, H1755 and DFC-1032 were used as target cells and were co-cultured with MAGE-B2CTL clones at different E:T ratios and cytotoxicity was measured. Cytotoxicity of MAGE-B2CTL clones was observed against lung cancer cell lines DFC-1032 and H1755 (FIG. 3C). Finally, cytotoxicity of CTL clones against the parental lung cancer cell lines PC-9 and H1573 and two cell lines with forced expression of HLA-A2 was assessed. Greater cytotoxic activity was observed against cell lines with forced expression of HLA-A2 compared to the parental PC-9 and H1573 cells (fig. 3D).

To generate MAGE-B2 TCR engineered T cells (TCR-T), TCR from MAGE-B2CTL clones were cloned and inserted into retroviral vector pMSGV 1. A linker fragment comprising a furin cleavage site, an SGSG linker and a P2A cleavage site was inserted between the TCR- β and TCR- α chains to ensure that both chains are expressed equally under the MSCV promoter. Recombinant retroviruses were produced by co-transfection of the retroviral vector and the envelope vector RD114 into the packaging cell line GP 2-293. Two to three days after transfection, the supernatant containing the retrovirus was used to infect allogeneic PBMC activated for 2 days at 50ng/mg OKT3 and 300U/ml IL-2 stimulation. Infection is performed once again after one day of the first infection. After 5 days clear CD8 was detected by flow cytometry ⁺ Tetramer ⁺ Group (fig. 4). Amplification of CD8 by sorting and using a rapid amplification protocol ⁺ Tetramer ⁺ The population was used to develop TCR-T cell lines.

Peptide titration assays were performed with T2 cells pulsed with varying concentrations of MAGE-B2 peptide (10 pg/ml to 10. Mu.g/ml) as target cells. T2 cells were co-cultured with MAGE-B2TCR-T cell lines (E: T=20:1). By usingStandard of ⁵¹ Cytotoxicity was detected by Cr release assay (fig. 5A). Cytotoxicity of MAGE-B2TCR-T against lung cancer cell line H2023 (HLA-A x 0201) and normal lung cell line HSAEC2-KT (HLA-A x 0201) was also assessed (fig. 5B). Lung cancer cell line H2023 and normal lung cell line HSAEC2-KT were co-cultured with MAGE-B2TCR-T cells at different e:t ratios. By standard ⁵¹ Cr release assay detects killing activity. The MAGE-B2TCR-T cell line was observed to be specific for cytotoxicity against lung cancer cell lines.

Finally, MAGE-B2TCR-T cells were functionally characterized by Intracellular Cytokine Staining (ICS). The MAGE-B2TCR-T cell line was co-cultured with lung cancer cell line H2023, normal lung cell line HSAEC2-KT, T2 pulsed with MAGE-B2 peptide and T2 pulsed with MART-1 peptide M26. IFN-gamma, TNF-alpha, IL-2 and antigen specific response markers CD137 and CD69 were detected by ICS assay. After co-culture, the levels of IFN-gamma, TNF-alpha, IL-2, CD137 and CD69 were significantly increased in the MAGE-B2TCR-T cell line when the TCR-T cells were co-cultured with the lung cancer cell line H2023 or T2 pulsed with the MAGE-B2 peptide, as compared to the normal lung cell line HSAEC2-KT or T2 co-cultured pulsed with the control peptide M26.

Thus, MAGE-B2 TCR-T cells can be used to treat HLA-A2 (e.g., HLA-A 0201, HLA-A 0202, HLA-A 0203, HLA-A 0204, or HLA-A 0205) positive patients with advanced or recurrent cancer, for example, by using allogeneic PBMCs to generate and amplify TCR gene modified CTLs. Following functional assays (e.g., phenotypes and cytotoxicity), TCR-modified T cells are infused into a patient.

Example 2 materials and methods

Generation of T cell clones:full length MAGE-B2 RNA was transfected into mature Dendritic Cells (DCs) derived from HLA-A2 healthy donors. RNA transfected DCs were co-cultured with naive T cells in the presence of IL-21 at a ratio of DC:T=1:10. After one week, T cells were re-stimulated with RNA transfected DCs. After two rounds of stimulation, CD8 and tetrameric double positive T cell populations were sorted and expanded using a Rapid Expansion Protocol (REP). T cell clones were generated by limiting dilution. Screening for high activity CT by cytotoxicity assay against cancer cellsCloning L.

T Cell Receptor (TCR) cloning and retroviral expression vector construction:TCR (comprising alpha and beta chains) were cloned using the 5'-RACE method according to the manufacturer's instructions. TCR V-alpha and TCRV-beta use was identified using the IMGT/V-QUEST annotation tool. For TCR expression retroviral vector construction, forward primers were designed based on TCR V- α or β use. Reverse primers were designed based on the sequence of the TCR α or β constant regions. An expression cassette comprising A-And β -TCR chains separated by furin and a P2A linker peptide was generated and the full length PCR product was cloned into retroviral vector pMSGV 1. The cloned DNA sequence was verified by sequencing.

Retrovirus production and infection of human Peripheral Blood Lymphocytes (PBLs):the pMSGV1 vector comprising TCR and the envelope vector RD114 were co-transfected into the packaging cell line GP2-293. After 6 to 8 hours of transfection, the medium was refreshed. The supernatant was harvested after 24 hours and added to a 6-well plate that had been coated with 20. Mu.g/mL retroNectin, followed by centrifugation (2000 Xg) at 32℃for 2 hours. The supernatant was then removed and PBL activated with 50ng/ml OKT3 and 300U/ml IL-2 for 2 days was added to the retroviral-loaded plates followed by centrifugation (1000 Xg) at 32℃for 10 minutes. Cells were then incubated overnight at 32 ℃ and the procedure repeated the next day (total of two transduction). After this, the cells were expanded at 37 ℃ in a 5% co2 incubator and split (split) as required.

Production of TCR-engineered T cell clones:after infection, on CD8 ⁺ And tetramer ⁺ T cell populations were sorted and expanded using the Rapid Expansion Protocol (REP).

⁵¹ Cr release measurement:using standard ⁵¹ Cr release assay to measure the killing ability of TCR engineered T cells or CTL clones to lyse HLA-A2 tumor targets. Tumor cells or normal cells were treated with 200. Mu. Ci at 37℃ ⁵¹ Cr is marked for 2 hours. The labeled target cells were washed and then incubated with effector cells in different ratios in 0.2ml complete medium at 37 ℃ for 4 hours. The harvested supernatant was counted using an automatic gamma counter.Maximum and spontaneous were determined by incubating the labeled target cells in trypan (trypan) lysis buffer or medium for 4 hours at 37 deg.c ⁵¹ Cr is released. Each data point was determined as the average of four wells in a row. Specific lysis was calculated as follows: killing% = ((specific release-spontaneous release)/(total release-spontaneous release)) ×100.

Intracellular Cytokine Staining (ICS) assay:t cells were incubated with target cells at a ratio of 10:1 overnight at 37℃in the presence of brefeldin A (BFA). After co-culture, T cells were harvested and washed. Cells were first stained with a flow antibody anti-surface marker. After this, the cells are washed and fixed with a fixing buffer, and then permeabilized using a permeabilization solution. The permeabilized cells are then stained with an intracellular cytokine flow antibody. Finally, FACS was used to analyze the level of cytokine production in cells.

Example 3 production of MAGE-B2 HLA-A2 restriction peptide (MB 2-231) specific TCR-T production

Dendritic cells pulsed with MAGE-B2 peptide (GVYDGEEHSV; SEQ ID NO: 1) were used to stimulate PBMC derived from the same healthy donor to produce additional MAGE-B2 specific T cell products (FIG. 7). After 2 stimulations, small cd8+/tetramers were observed in 3 wells of one 48-well plate ⁺ A group. The 3 positive wells were separately sorted using tetramer-directed sorting techniques and amplified with REP for 1 or 2 rounds. The CD8 and tetramer staining of the final product is shown in figure 7.

The functional avidity of 3 MAGE-B2 specific CTL cell lines was shown by lysis of T2 cell lines pulsed with various concentrations of MAGE-B2 peptide (GVYDGEEHSV; SEQ ID NO: 1) at a 20:1 effector to target (E: T) ratio. Cytotoxicity was detected using a standard 51Cr release assay (fig. 8A). 3 MAGE-B2 specific CTL cell lines were also evaluated against lung cancer cell line H2023 (HLA-A x 0201 ⁺ ，MAGE-B2 ⁺ ) And the normal lung cell line HSAEC2-KT (HLA-A x 0201 ⁺ Cytotoxicity of MAGE-B2-). Lung cancer cell line H2023 and normal lung cell line HSAEC2-KT were co-cultured with MAGE-B2 TCR-T cells at different e:t ratios. By standard 51Cr release assay the killing activity was measured. All 3 MAGE-B2 specific CTL cell lines were observed to have specific cytotoxicity against lung cancer cell line H2023 (fig. 8B). Cytotoxicity of 3 MAGE-B2 specific CTL cell lines against other lung cancer cell lines H1299 (HLA-A 0201-, MAGE-b2+), H1299-A2 (HLA-A 0201), MAGE-b2+), H1395 (HLA-A 0201+, MAGE-b2+), H522 (HLA-A 0201+, MAGE-B2-), H1355 (HLA-A 0201+, MAGE-B2-), H1755 (HLA-A 0201+, MAGE-B2-), MAGE-B2+), and DFC-1032 (HLA-A 0201+, MAGE-B2-) (fig. 8C, 8D, 8E) was also assessed.

To generate MAGE-B2TCR engineered T cells (TCR-T), TCR from MAGE-B2CTL cell line C5 was cloned and inserted into retroviral vector pMSGV 1. A linker fragment comprising a furin cleavage site, an SGSG linker and a P2A cleavage site was inserted between the TCR- β and TCR- α chains to ensure that both chains are expressed equally under the MSCV promoter. Recombinant retroviruses were produced by co-transfection of the retroviral vector and the envelope vector RD114 into the packaging cell line Phoenix-GP. Two to three days after transfection, the supernatant containing the retrovirus was used to infect PBMCs of allogeneic HLA-A 0201+ healthy donors that were activated for 2 days under 50ng/mg OKT3 and 300U/ml IL-2 stimulation. After 5 days, clear cd8+ tetramer+ populations were detected by flow cytometry (fig. 9A). Cd8+ tetramer+ populations were sorted using tetramer-directed sorting techniques and amplified with REP. CD8 and tetramer staining of the final product is shown in fig. 9A. The functional avidity of MAGE-B2TCR-T was shown by lysis of T2 cell lines pulsed with various concentrations of MAGE-B2 peptide (GVYDGEEHSV; SEQ ID NO: 1) at a 20:1 effector to target (E: T) ratio. Cytotoxicity was detected using a standard 51Cr release assay (fig. 9B). Cytotoxicity of MAGE-B2TCR-T against lung cancer cell line H2023 (HLA-A 0201+, MAGE-b2+) and normal lung cell line HSAEC2-KT (HLA-A 0201+, MAGE-B2-) was also assessed (fig. 9C). Cytotoxicity of MAGE-B2TCR-T against other lung cancer cell lines H1299 (HLA-A x 0201-, MAGE-b2+), H1299-A2 (HLA-A x 0201) forced expression, MAGE-b2+), H1395 (HLA-A x 0201+, MAGE-b2+), H522 (HLA-A x 0201+, MAGE-B2-), H1355 (HLA-A x 0201+, MAGE-B2-), H1755 (HLA-A x 0201+, MAGE-b2+) and DFC-1032 (HLA-A x 0201+, MAGE-B2-) (9D, 9E) was also assessed.

Finally, MAGE-B2 TCR-T cells were functionally characterized by Intracellular Cytokine Staining (ICS). MAGE-B2 TCR-T cell lines were co-cultured with T2 pulsed with MAGE-B2 peptide (GVYDGEEHSV; SEQ ID NO: 1) and T2 pulsed with MART-1 peptide M26 (as a control) (FIG. 10A). The response of MAGE-B2 specific TCR-T to lung cancer cell line H2023, normal lung cell line HSAEC2-KT (as a control) was also assessed (FIG. 10A). In addition, other lung cancer cell lines H1395, H522, H1299-A2, DFC-1032, H1355 and H1755 were also used as targets for evaluating the function and specificity of MAGE-B2 TCR-T (FIGS. 10B, 10C). The up-regulation of IFN-gamma, TNF-alpha cytokine release and antigen specific response markers CD137 and CD69 of MAGE-B2 TCR-T cell lines was examined with ICS.

***

In light of this disclosure, all methods disclosed and claimed herein can be made and executed without undue experimentation. While the compositions and methods of this invention have been described in terms of several preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Reference to the literature

The following references are specifically incorporated herein by reference to the extent that they provide exemplary operations or other details that supplement the disclosure set forth herein.

Barnea et al.，Eur J Immunol，32(1)：213-22，2002.

Remington：The Science and Practice of Pharmacy，22nd Edition，Pharmaceutical Press，2012.

Shraibman et al.，Mol Cell Proteomics，15(9)：3058-70，2016.

Sequence listing

<110> Board of Texas university System board

<120> T cell receptor with MAGE-B2 specificity and uses thereof

<130> UTFC.P1372WO

<150> 62/660,083

<151> 2018-04-19

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<223> alpha chain (TRAV 10. Times.01)

<400> 18

atgaaaaagc atctgacgac cttcttggtg attttgtggc tttattttta tagggggaat 60

ggcaaaaacc aagtggagca gagtcctcag tccctgatca tcctggaggg aaagaactgc 120

actcttcaat gcaattatac agtgagcccc ttcagcaact taaggtggta taagcaagat 180

actgggagag gtcctgtttc cctgacaatc atgactttca gtgagaacac aaagtcgaac 240

ggaagatata cagcaactct ggatgcagac acaaagcaaa gctctctgca catcacagcc 300

tcccagctca gcgattcagc ctcctacatc tgtgtggtga tttcaggctt tcagaaactt 360

gtatttggaa ctggcacccg acttctggtc agtccaaata tccagaaccc tgaccctgcc 420

gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 480

gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 540

gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 600

tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 660

cccagcccag aaagttcctg tgatgtcaag ctggtcgaga aaagctttga aacagatacg 720

aacctaaact ttcaaaacct gtcagtgatt gggttccgaa tcctcctcct gaaagtggcc 780

gggtttaatc tgctcatgac gctgcggctg tggtccagct aa 822

<210> 19

<211> 273

<212> PRT

<213> artificial sequence

<220>

<223> alpha chain

<400> 19

Met Lys Lys His Leu Thr Thr Phe Leu Val Ile Leu Trp Leu Tyr Phe

1 5 10 15

Tyr Arg Gly Asn Gly Lys Asn Gln Val Glu Gln Ser Pro Gln Ser Leu

20 25 30

Ile Ile Leu Glu Gly Lys Asn Cys Thr Leu Gln Cys Asn Tyr Thr Val

35 40 45

Ser Pro Phe Ser Asn Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly

50 55 60

Pro Val Ser Leu Thr Ile Met Thr Phe Ser Glu Asn Thr Lys Ser Asn

65 70 75 80

Gly Arg Tyr Thr Ala Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu

85 90 95

His Ile Thr Ala Ser Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val

100 105 110

Val Ile Ser Gly Phe Gln Lys Leu Val Phe Gly Thr Gly Thr Arg Leu

115 120 125

Leu Val Ser Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu

130 135 140

Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe

145 150 155 160

Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile

165 170 175

Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn

180 185 190

Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala

195 200 205

Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu

210 215 220

Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr

225 230 235 240

Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu

245 250 255

Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser

260 265 270

Ser

<210> 20

<211> 939

<212> DNA

<213> artificial sequence

<220>

<223> beta chain (TRBV 11-3 x 04)

<400> 20

atgggtacca ggctcctctg ctgggtggcc ttctgtctcc tggtggaaga actcatagaa 60

gctggagtgg ttcagtctcc cagatataag attatagaga aaaaacagcc tgtggctttt 120

tggtgcaatc ctatttctgg ccacaatacc ctttactggt accggcagaa cttgggacag 180

ggcccggagc ttctgattcg atatgagaat gaggaagcag tagacgattc acagttgcct 240

aaggatcgat tttctgcaga gaggctcaaa ggagtagact ccactctcaa gatccagcct 300

gcagagcttg gggactcggc cgtgtatctc tgtgccagca gcttccctaa acagggatcc 360

tacaatgagc agttcttcgg gccagggaca cggctcaccg tgctagagga cctgaaaaac 420

gtgttcccac ccgaggtcgc tgtgtttgag ccatcagaag cagagatctc ccacacccaa 480

aaggccacac tggtgtgcct ggccacaggc ttcttccctg accacgtgga gctgagctgg 540

tgggtgaatg ggaaggaggt gcacagtggg gtcagcacgg acccgcagcc cctcaaggag 600

cagcccgccc tcaatgactc cagatactgc ctgagcagcc gcctgagggt ctcggccacc 660

ttctggcaga acccccgcaa ccacttccgc tgtcaagtcc agttctacgg gctctcggag 720

aatgacgagt ggacccagga tagggccaaa cccgtcaccc agatcgtcag cgccgaggcc 780

tggggtagag cagactgtgg ctttacctcg gtgtcctacc agcaaggggt cctgtctgcc 840

accatcctct atgagatcct gctagggaag gccaccctgt atgctgtgct ggtcagcgcc 900

cttgtgttga tggccatggt caagagaaag gatttctaa 939

<210> 21

<211> 312

<212> PRT

<213> artificial sequence

<220>

<223> beta chain

<400> 21

Met Gly Thr Arg Leu Leu Cys Trp Val Ala Phe Cys Leu Leu Val Glu

1 5 10 15

Glu Leu Ile Glu Ala Gly Val Val Gln Ser Pro Arg Tyr Lys Ile Ile

20 25 30

Glu Lys Lys Gln Pro Val Ala Phe Trp Cys Asn Pro Ile Ser Gly His

35 40 45

Asn Thr Leu Tyr Trp Tyr Arg Gln Asn Leu Gly Gln Gly Pro Glu Leu

50 55 60

Leu Ile Arg Tyr Glu Asn Glu Glu Ala Val Asp Asp Ser Gln Leu Pro

65 70 75 80

Lys Asp Arg Phe Ser Ala Glu Arg Leu Lys Gly Val Asp Ser Thr Leu

85 90 95

Lys Ile Gln Pro Ala Glu Leu Gly Asp Ser Ala Val Tyr Leu Cys Ala

100 105 110

Ser Ser Phe Pro Lys Gln Gly Ser Tyr Asn Glu Gln Phe Phe Gly Pro

115 120 125

Gly Thr Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro

130 135 140

Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln

145 150 155 160

Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val

165 170 175

Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser

180 185 190

Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg

195 200 205

Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn

210 215 220

Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu

225 230 235 240

Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val

245 250 255

Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser

260 265 270

Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu

275 280 285

Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met

290 295 300

Ala Met Val Lys Arg Lys Asp Phe

305 310

<210> 22

<211> 18

<212> DNA

<213> artificial sequence

<220>

<223> alpha chain CDR1

<400> 22

gtgagcccct tcagcaac 18

<210> 23

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> alpha chain CDR1

<400> 23

Val Ser Pro Phe Ser Asn

1 5

<210> 24

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> alpha chain CDR2

<400> 24

atgactttca gtgagaacac a 21

<210> 25

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> alpha chain CDR2

<400> 25

Met Thr Phe Ser Glu Asn Thr

1 5

<210> 26

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> alpha chain CDR3

<400> 26

gtggtgattt caggctttca gaaacttgta 30

<210> 27

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> alpha chain CDR3

<400> 27

Val Val Ile Ser Gly Phe Gln Lys Leu Val

1 5 10

<210> 28

<211> 15

<212> DNA

<213> artificial sequence

<220>

<223> beta chain CDR1

<400> 28

tctggccaca atacc 15

<210> 29

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> beta chain CDR1

<400> 29

Ser Gly His Asn Thr

1 5

<210> 30

<211> 18

<212> DNA

<213> artificial sequence

<220>

<223> beta chain CDR2

<400> 30

tatgagaatg aggaagca 18

<210> 31

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> beta chain CDR2

<400> 31

Tyr Glu Asn Glu Glu Ala

1 5

<210> 32

<211> 42

<212> DNA

<213> artificial sequence

<220>

<223> beta chain CDR3

<400> 32

gccagcagct tccctaaaca gggatcctac aatgagcagt tc 42

<210> 33

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> beta chain CDR3

<400> 33

Ala Ser Ser Phe Pro Lys Gln Gly Ser Tyr Asn Glu Gln Phe

1 5 10

<210> 34

<211> 57

<212> DNA

<213> artificial sequence

<220>

<223> alpha chain Signal peptide

<400> 34

atgaactatt ctccaggctt agtatctctg atactcttac tgcttggaag aacccgt 57

<210> 35

<211> 19

<212> PRT

<213> artificial sequence

<220>

<223> alpha chain Signal peptide 3

<400> 35

Met Asn Tyr Ser Pro Gly Leu Val Ser Leu Ile Leu Leu Leu Leu Gly

1 5 10 15

Arg Thr Arg

<210> 36

<211> 336

<212> DNA

<213> artificial sequence

<220>

<223> alpha chain variable region 2

<400> 36

ggaaattcag tgacccagat ggaagggcca gtgactctct cagaagaggc cttcctgact 60

ataaactgca cgtacacagc cacaggatac ccttcccttt tctggtatgt ccaatatcct 120

ggagaaggtc tacagctcct cctgaaagcc acgaaggctg atgacaaggg aagcaacaaa 180

ggttttgaag ccacataccg taaagaaacc acttctttcc acttggagaa aggctcagtt 240

caagtgtcag actcagcggt gtacttctgt gctctgacca acgactacaa gctcagcttt 300

ggagccggaa ccacagtaac tgtaagagca aatatc 336

<210> 37

<211> 111

<212> PRT

<213> artificial sequence

<220>

<223> alpha chain variable region 3

<400> 37

Gly Asn Ser Val Thr Gln Met Glu Gly Pro Val Thr Leu Ser Glu Glu

1 5 10 15

Ala Phe Leu Thr Ile Asn Cys Thr Tyr Thr Ala Thr Gly Tyr Pro Ser

20 25 30

Leu Phe Trp Tyr Val Gln Tyr Pro Gly Glu Gly Leu Gln Leu Leu Leu

35 40 45

Lys Ala Thr Lys Ala Asp Asp Lys Gly Ser Asn Lys Gly Phe Glu Ala

50 55 60

Thr Tyr Arg Lys Glu Thr Thr Ser Phe His Leu Glu Lys Gly Ser Val

65 70 75 80

Gln Val Ser Asp Ser Ala Val Tyr Phe Cys Ala Leu Thr Asn Asp Tyr

85 90 95

Lys Leu Ser Phe Gly Ala Gly Thr Thr Val Thr Val Arg Ala Asn

100 105 110

<210> 38

<211> 420

<212> DNA

<213> artificial sequence

<220>

<223> alpha chain constant region 2

<400> 38

cagaaccctg accctgccgt gtaccagctg agagactcta aatccagtga caagtctgtc 60

tgcctattca ccgattttga ttctcaaaca aatgtgtcac aaagtaagga ttctgatgtg 120

tatatcacag acaaaactgt gctagacatg aggtctatgg acttcaagag caacagtgct 180

gtggcctgga gcaacaaatc tgactttgca tgtgcaaacg ccttcaacaa cagcattatt 240

ccagaagaca ccttcttccc cagcccagaa agttcctgtg atgtcaagct ggtcgagaaa 300

agctttgaaa cagatacgaa cctaaacttt caaaacctgt cagtgattgg gttccgaatc 360

ctcctcctga aagtggccgg gtttaatctg ctcatgacgc tgcggctgtg gtccagctga 420

<210> 39

<211> 140

<212> PRT

<213> artificial sequence

<220>

<223> alpha chain constant region 3

<400> 39

Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser

1 5 10 15

Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn

20 25 30

Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val

35 40 45

Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp

50 55 60

Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile

65 70 75 80

Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val

85 90 95

Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln

100 105 110

Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly

115 120 125

Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

130 135 140

<210> 40

<211> 57

<212> DNA

<213> artificial sequence

<220>

<223> beta chain Signal peptide 4

<400> 40

atgggtcctg ggcttctcca ctggatggcc ctttgtctcc ttggaacagg tcatggg 57

<210> 41

<211> 19

<212> PRT

<213> artificial sequence

<220>

<223> beta chain Signal peptide 5

<400> 41

Met Gly Pro Gly Leu Leu His Trp Met Ala Leu Cys Leu Leu Gly Thr

1 5 10 15

Gly His Gly

<210> 42

<211> 342

<212> DNA

<213> artificial sequence

<220>

<223> beta chain variable region 4

<400> 42

gatgccatgg tcatccagaa cccaagatac caggttaccc agtttggaaa gccagtgacc 60

ctgagttgtt ctcagacttt gaaccataac gtcatgtact ggtaccagca gaagtcaagt 120

caggccccaa agctgctgtt ccactactat gacaaagatt ttaacaatga agcagacacc 180

cctgataact tccaatccag gaggccgaac acttctttct gctttcttga catccgctca 240

ccaggcctgg gggacgcagc catgtacctg tgtgccacca gcaggggcgg gaggtacaat 300

gagcagttct tcgggccagg gacacggctc accgtgctag ag 342

<210> 43

<211> 114

<212> PRT

<213> artificial sequence

<220>

<223> beta chain variable region 5

<400> 43

Asp Ala Met Val Ile Gln Asn Pro Arg Tyr Gln Val Thr Gln Phe Gly

1 5 10 15

Lys Pro Val Thr Leu Ser Cys Ser Gln Thr Leu Asn His Asn Val Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Ser Ser Gln Ala Pro Lys Leu Leu Phe His

35 40 45

Tyr Tyr Asp Lys Asp Phe Asn Asn Glu Ala Asp Thr Pro Asp Asn Phe

50 55 60

Gln Ser Arg Arg Pro Asn Thr Ser Phe Cys Phe Leu Asp Ile Arg Ser

65 70 75 80

Pro Gly Leu Gly Asp Ala Ala Met Tyr Leu Cys Ala Thr Ser Arg Gly

85 90 95

Gly Arg Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val

100 105 110

Leu Glu

<210> 44

<211> 531

<212> DNA

<213> artificial sequence

<220>

<223> beta strand constant region 4

<400> 44

gacctgaaaa acgtgttccc acccgaggtc gctgtgtttg agccatcaga agcagagatc 60

tcccacaccc aaaaggccac actggtgtgc ctggccacag gcttcttccc tgaccacgtg 120

gagctgagct ggtgggtgaa tgggaaggag gtgcacagtg gggtcagcac ggacccgcag 180

cccctcaagg agcagcccgc cctcaatgac tccagatact gcctgagcag ccgcctgagg 240

gtctcggcca ccttctggca gaacccccgc aaccacttcc gctgtcaagt ccagttctac 300

gggctctcgg agaatgacga gtggacccag gatagggcca aacccgtcac ccagatcgtc 360

agcgccgagg cctggggtag agcagactgt ggctttacct cggtgtccta ccagcaaggg 420

gtcctgtctg ccaccatcct ctatgagatc ctgctaggga aggccaccct gtatgctgtg 480

ctggtcagcg cccttgtgtt gatggccatg gtcaagagaa aggatttctg a 531

<210> 45

<211> 175

<212> PRT

<213> artificial sequence

<220>

<223> beta strand constant region 5

<400> 45

Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser

1 5 10 15

Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala

20 25 30

Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly

35 40 45

Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys Glu

50 55 60

Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu Arg

65 70 75 80

Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln

85 90 95

Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg

100 105 110

Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala

115 120 125

Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly Val Leu Ser Ala

130 135 140

Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val

145 150 155 160

Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp

165 170 175

Claims (53)

1. An isolated T Cell Receptor (TCR) capable of binding an antigenic peptide derived from melanomA-Associated antigen B2 (MAGE-B2), comprising a TCR a polypeptide of SEQ ID No. 3 and a TCR β polypeptide of SEQ ID No. 5; or a TCR alpha polypeptide comprising SEQ ID NO. 19 and a TCR beta polypeptide comprising SEQ ID NO. 21.

2. The TCR of claim 1, wherein the antigenic peptide is HLA-A2 restricted.

3. The TCR of claim 2, wherein the antigenic peptide is HLA-A x 0201-restricted.

4. The TCR of claim 1, wherein the TCR a polypeptide comprises CDR1 of sequence SEQ ID No. 7, CDR2 of sequence SEQ ID No. 9, and CDR3 of sequence SEQ ID No. 11, and the TCR β polypeptide comprises CDR1 of sequence SEQ ID No. 13, CDR2 of sequence SEQ ID No. 15, and CDR3 of sequence SEQ ID No. 17.

5. The TCR of claim 1, wherein the TCR a polypeptide comprises CDR1 of sequence SEQ ID No. 23, CDR2 of sequence SEQ ID No. 25, and CDR3 of sequence SEQ ID No. 27, and the TCR β polypeptide comprises CDR1 of sequence SEQ ID No. 29, CDR2 of sequence SEQ ID No. 31, and CDR3 of sequence SEQ ID No. 33.

6. The TCR of claim 1, wherein the TCR is a soluble TCR lacking a transmembrane domain.

7. The TCR of claim 6, further comprising a detectable label.

8. The TCR of any one of claim 6 or claim 7, further comprising a therapeutic agent.

9. A multivalent TCR complex comprising a plurality of TCRs according to any one of claims 1 to 8.

10. The complex of claim 9, wherein the multivalent TCR comprises 2, 3, 4, or more TCRs.

11. The complex of claim 10, wherein the multivalent TCR is present in a lipid bilayer or attached to a particle.

12. The complex of claim 10, wherein the TCR is conjugated via a linker molecule.

13. A polypeptide comprising a TCR alpha polypeptide comprising CDR1 as set forth in sequence SEQ ID No. 7, CDR2 as set forth in sequence SEQ ID No. 9, and CDR3 as set forth in sequence SEQ ID No. 11, and a TCR beta polypeptide comprising CDR1 as set forth in sequence SEQ ID No. 13, CDR2 as set forth in sequence SEQ ID No. 15, and CDR3 as set forth in sequence SEQ ID No. 17.

14. A polypeptide comprising a TCR alpha polypeptide comprising CDR1 as set forth in sequence SEQ ID No. 23, CDR2 as set forth in SEQ ID No. 25, and CDR3 as set forth in SEQ ID No. 27, and a TCR beta polypeptide comprising CDR1 as set forth in sequence SEQ ID No. 29, CDR2 as set forth in SEQ ID No. 31, and CDR3 as set forth in SEQ ID No. 33.

15. A polynucleotide encoding the polypeptide of any one of claims 13 to 14.

16. An expression vector comprising a TCR as claimed in any one of claims 1 to 8.

17. The expression vector of claim 16, wherein the expression vector is a viral vector.

18. The expression vector of claim 17, wherein the viral vector is a retroviral vector or a lentiviral vector.

19. The expression vector of any one of claims 16 to 18, further comprising a linker domain.

20. The expression vector of claim 19, wherein the linker domain is between the tcra polypeptide and tcrp polypeptide.

21. The expression vector of claim 19, wherein the linker domain comprises one or more cleavage sites.

22. The expression vector of claim 21, wherein the one or more cleavage sites are furin cleavage sites and/or P2A cleavage sites.

23. The expression vector of claim 21, wherein the one or more cleavage sites are separated by a spacer.

24. The expression vector of claim 23, wherein the spacer is SGSG or GSG.

25. The expression vector of claim 19, wherein the tcra polypeptide and tcrp polypeptide are linked by an IRES sequence.

26. A host cell engineered to express a TCR according to any one of claims 1 to 8, wherein the host cell is engineered to be a MAGE-B2 specific cell.

27. The host cell of claim 26, wherein the cell is an immune cell.

28. The host cell of claim 26, wherein the cell is an NK cell, a constant NK cell, a NKT cell, a Mesenchymal Stem Cell (MSC), or an Induced Pluripotent Stem (iPS) cell.

29. The host cell of claim 26, wherein the cell is isolated from umbilical cord or blood.

30. The host cell of claim 26, wherein the immune cell is a T cell or a peripheral blood lymphocyte.

31. The host cell of claim 30, wherein the T cell is CD8 ⁺ T cells, cd4+ T cells or γδ T cells.

32. The host cell of claim 30, wherein the cell is allogeneic or autologous.

33. A pharmaceutical composition comprising the MAGE-B2 specific cell population according to any one of claims 26 to 32.

34. A method for engineering a MAGE-B2 specific immune cell comprising contacting the immune cell with the expression vector of any one of claims 16 to 24.

35. The method of claim 34, wherein the immune cell is a T cell, a peripheral blood lymphocyte, an NK cell, a constant NK cell, or a NKT cell.

36. The method of claim 34 or claim 35, wherein contacting is further defined as transfection or transduction.

37. The method of claim 35, wherein the peripheral blood lymphocytes are stimulated with OKT3 and IL-2.

38. The method of claim 34, further comprising sorting the immune cells to isolate TCR-engineered T cells, T cell cloning by serial dilution, and expansion of T cell clones by a rapid expansion protocol.

39. A composition comprising a therapeutically effective amount of a MAGE-B2 specific cell according to any one of claims 26 to 32 for use in treating cancer in a subject.

40. The composition of claim 39, wherein said MAGE-B2 specific cells are T cells.

41. Use of a MAGE-B2 specific cell according to any one of claims 26 to 32 in the manufacture of a medicament for treating cancer in a subject, said treatment comprising administering to said subject a therapeutically effective amount of a MAGE-B2 specific cell according to any one of claims 26 to 32.

42. The use of claim 41, wherein said MAGE-B2 specific cell is a T cell.

43. The use of claim 41, wherein the subject is identified as having HLA-A 0201, HLA-A 0202, HLA-A 0203, HLA-A 0204, or HLA-A 0205 alleles.

44. The use of claim 41, further comprising the step of lymphocyte depletion of said subject prior to administration of said therapeutically effective amount of MAGE-B2 specific T cells.

45. The use of claim 42, wherein the therapeutically effective amount of MAGE-B2 specific T cells is derived from a sample of autologous Tumor Infiltrating Lymphocytes (TILs) having anti-tumor activity.

46. The use of claim 41, wherein said MAGE-B2 specific cells are administered intravenously, intraperitoneally, or intratumorally to said subject.

47. The use of claim 41, wherein the subject is a human.

48. The use of claim 41, wherein said treatment further comprises the step of administering at least one additional therapeutic agent to said subject.

49. The use of claim 48, wherein said at least one additional therapeutic agent is selected from the group consisting of chemotherapy, radiation therapy, and immunotherapy.

50. The use of claim 48, wherein said at least one additional therapeutic agent is immunotherapy.

51. The use of claim 50, wherein the immunotherapy is an immune checkpoint inhibitor.

52. The use of claim 51, wherein the immune checkpoint inhibitor inhibits an immune checkpoint protein or ligand thereof selected from the group consisting of: CTLA-4, PD-1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR or adenosine A2a receptor (A2 aR).

53. The use of claim 52, wherein the immune checkpoint inhibitor inhibits PD-1 or CTLA-4.